Combined cytogenetic, somatic cell genetic, immunologic, and molecular methods will be used to clarify the role of gene amplification in carcinogenesis and tumor progression. Attention will be focused on mammalian ribosomal RNA genes, in which amplification is associated with hypermethylation and transcriptional inactivity. We will attempt to activate them by demethylation with 5-azacytidine in order to establish if DNA methylation has a role in rRNA gene regulation. Restriction mapping of the rDNA of inactive and reactivated genes may identify any sites of methylation involved in rRNA gene regulation. The relationship between amplification and hypermethylation of rRNA genes will be studied in various ways, e.g., by amplifying active, nonmethylated genes by co-transformation with cloned rRNA and DHFR sequences. Results thus far indicate that independent cloned fragments of mouse rDNA and DHFR sequences can be co-integrated and co-amplified in Chinese hamster cells by selecting for higher levels. Similarly, a cloned fragment of human rDNA that has been placed (in each orientation) adjacent to an engineered DHFR gene within a plasmid can be co-amplified in hamster cells. S1 nuclease protection assays using the species-specific external transcribed spacer regions suggest that the amplified mouse rDNA is transcribed and that the amplified human rDNA in one orientation is also transcribed in the hamster cells. Confirmation will be sought and the hypothesis explored that species-specific protein co-factors are not required for rRNA transcription when the rDNA is adjacent to a sequence with enhancer activity. Finally, the nature of the amplified sequences in untreated tumor cells will be studied by molecular cloning. (A)